Bacterial cell wall biosynthesis is one of the major targets where many antibiotics are designed and acted upon. The cell walls of both Gram-positive and -negative bacteria consist of layers of peptidoglycan, which has a mesh-like structure scaffolding the cytoplasmic membrane. Cell wall maintains the shape and integrity of bacteria. It can protect bacterial cells against osmotic pressure, and its disruption can lead to cell lysis and death. (Holtje, J. V. (1998). Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol Mol Biol Rev 62, 181-203.)
The discovery and clinical development of penicillin ushered in the modern antibiotic era and stimulated the discovery of the antibiotics in current clinical use. Some 80 years after their discovery, penicillins and related antibiotics (collectively called β-lactams) remain clinically useful. Nevertheless, the remarkable ability of bacteria to develop resistance to β-lactam and other antibiotics means that there is a continued need for new antibiotic targets and new antimicrobial agents. (Wright G D. Science (2007) 315(5817):1373-1374.)
Penicillin and other β-lactam antibiotics target several bacterial enzymes, collectively termed penicillin-binding proteins (PBPs). PBPs are necessary for the growth and maintenance of the peptidoglycan layer, which forms part of the bacterial cell wall and protects the cell from osmotic stress. Inhibition of peptidoglycan biosynthesis and of its controlled breakdown (for example, to enable partition of the cell wall during cell division) therefore inhibits cell growth. Because the peptidoglycan polymer is ubiquitous and essential to bacterial life, its assembly and maintenance are targets for many antibiotics.
Bacteria use a peptidoglycan layer to protect themselves from osmotic stress. Synthesis of this layer proceeds in several steps. First, lipid II is synthesized in the cell. It is then transferred to the outside, where it is added to the peptidoglycan polymer by membrane-associated transglycosylase enzymes. Finally, the polymer is cross-linked via interstrand peptide bonds catalyzed by transpeptidase enzymes.
The peptidoglycan consists of a backbone chain of repeating two-sugar units (called NAG and NAM) and a pentapeptide chain bound to each NAM. The NAG-NAM-pentapeptide core (called lipid II) is synthesized in the cell and tethered to the cell membrane by a lipid linker. Lipid II is then transferred from the inside of the cell to the outside, where membrane-associated glycosyltransferases assist in grafting it onto the polymer. Transpeptidases catalyze the formation of peptide bonds between polymer strands, thereby making the wall more rigid. These tasks are performed by bifunctional enzymes that contain glycosyltransferase and transpeptidase domains; the latter are sensitive to β-lactams.
The peptidoglycan glycosyltransferase activity of the bifunctional enzymes is an excellent target for the development of new antibiotics. The bifunctional enzymes include PBP1b from Escherichia coli. Despite their importance to bacterial physiology and drug discovery, they have resisted detailed study, mainly because these large membrane proteins are difficult to purify, assay, and crystallize.
The high-molecular-weight penicillin-binding proteins (PBPs) are responsible for the enlargement of the essential bacterial murein (peptidoglycan) sacculus by transpeptidation and transglycosylation of the murein precursors (Park, J. T. 1996. The murein sacculus, p. 48-57. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology. ASM Press, Washington, D.C.). In E. coli there are three bifunctional enzymes catalyzing both reactions, PBP1A, PBP1B, and PBP1C, and two monofunctional transpeptidases, PBP2 and PBP3. (Höltje, J.-V. 1998. Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol. Mol. Biol. Rev. 62:181-203). PBP1a and PBP 1b are the major bifunctional enzymes (Ishino, F., K. Mitsui, S. Tamaki, and M. Matsuhashi. 1980. Biochem. Biophys. Res. Commun. 97:287-293; Terrak, M., et al., 1999. Mol. Microbiol. 34:350-364.), and a deletion of both is lethal for the cell (Suzuki, H., Y. Nishimura, and Y. Hirota. 1978. On the process of cellular division in E. coli: a series of mutants of E. coli altered in the penicillin-binding proteins. Proc. Natl. Acad. Sci. USA 75:664-668). Encoded by a single gene (ponB or mrcB), PBP1b was shown to exist in three forms (α, β, and γ) which differ in the length of the short cytoplasmic part of the protein. (Nakagawa, J., and M. Matsuhashi. 1982. Molecular divergence of a major peptidoglycan synthetase with transglycosylase-transpeptidase activities in Escherichia coli-penicillin-binding protein 1Bs. Biochem. Biophys. Res. Commun. 105:1546-1553).
PBP1a and PBP1b are not essential for cell growth, but cells lacking both enzymes are not viable, indicating that both have a similar, essential function that cannot be taken over by other murein synthases (Suzuki, H., Nishimura, Y., and Hirota, Y. (1978) Proc. Natl. Acad. Sci. U.S.A 75, 664-668; Yousif, S. Y., Broome-Smith, J. K., and Spratt, B. G. (1985) J. Gen. Microbiol. 131, 2839-2845). Yet, mutants lacking either PBP1a or PBP1b show particular phenotypes, indicating that these synthases may play distinct roles during cell growth and division. For example, mutants without PBP1b are more sensitive to β-lactam antibiotics than mutants without PBP1a (Yousif, S. Y., Broome-Smith, J. K., and Spratt, B. G. (1985) J. Gen. Microbiol. 131, 2839-2845).
Escherichia coli PBP1b is a bifunctional transglycosylase, also known as peptidoglycan glycosyltransferase or murein synthase. It contains a transmembrane (TM) helix, two enzymatic domains—transglycosylase (TG) and transpeptidase (TP) (Goffin C, Ghuysen J M (1998). Microbiol Mol Biol Rev 62:1079-1093), and a domain composed of about 100 amino acid residues between TM and TG with unknown structure and functionality (FIG. 2B). For over 50 years, TP has been the main target for 2 most important classes of antibiotics: β-lactams (e.g., penicillin and methicillin) and glycopeptides (e.g., vancomycin). Not too long after they were introduced, resistant bacteria had emerged rapidly and caused serious medical problems. In contrast, resistant strains against moenomycin, the only natural inhibitor to TG from Streptomyces, have rarely been found. The development of new antibiotics against TG domains has been highly anticipated (Halliday J, McKeveney D, Muldoon C, Rajaratnam P, Meutermans W (2006) Biochem Pharmacol 71:957-967), and not until recently have the molecular structures of TG domain been available, even with the TM structure undefined.
During the years, resistance bacteria strains against two of the most important antibiotics, β-lactam (such as penicillin) and glycopeptide (such as vancomycin), have become a very serious medical problem in the treatment of bacterial infections. (Fisher, J. F., Meroueh, S. O. & Mobashery, S. (2005). Chem Rev 105, 395-424.; Pootoolal, J., Thomas, M. G., Marshall, C. G., Neu, J. M., Hubbard, B. K., Walsh, C. T. & Wright, G. D. (2002). Proc Natl Acad Sci USA 99, 8962-7.) β-lactam and glycopeptide antibiotics target against the transpeptidation process, i.e. the action of transpeptidase.
Unlike the prevalence of antibiotic resistant bacterial strains against transpeptidase, resistance phenotype against transglycosylase has not been reported. There is only one reported inhibitor against transglycosylase, Moenomycin. Therefore, there is a need for new antibiotics against the transglycosylase domain of PBP1b.